Search

To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.

It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA’s genome integrity. Cosmic radiation due to Earth’s weak magnetic field and alkylating metabolic radicals added to these threats. Here, we propose that ancient forms of life had only two distinct repair mechanisms: versatile apurinic/apyrimidinic (AP) endonucleases to cope with both AP sites and deaminated residues, and enzymes catalyzing the direct reversal of UV and alkylation damage. The absence of uracil–DNA N-glycosylases in some Archaea, together with the presence of an AP endonuclease, which can cleave uracil-containing DNA, suggests that the AP endonuclease-initiated nucleotide incision repair (NIR) pathway evolved independently from DNA glycosylase-mediated base excision repair. NIR may be a relic that appeared in an early thermophilic ancestor to counteract spontaneous DNA damage. We hypothesize that a rise in the oxygen level in the Earth’s atmosphere ~2 Ga triggered the narrow specialization of AP endonucleases and DNA glycosylases to cope efficiently with a widened array of oxidative base damage and complex DNA lesions.

International audience; Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyrimidinic (AP) endonuclease 1 (APE1/APEX1/HAP-1), referred also as a redox factor 1 (Ref-1), stimulates the DNA binding activities of the oxidized TFs such as AP-1 and NF-κB. Also, APE1 participates in the base excision repair (BER) and nucleotide incision repair (NIR) pathways to remove oxidative DNA base damage. At present, the molecular mechanism underlying the TF-stimulating/redox function of APE1 and its biological role remains disputed. Here, we provide evidence that, instead of direct cysteine reduction in TFs by APE1, APE1-catalyzed NIR and TF-stimulating activities may be based on transient cooperative binding of APE1 to DNA and induction of conformational changes in the helix. The structure of DNA duplex strongly influences NIR and TF-stimulating activities. Homologous plant AP endonucleases lacking conserved cysteine residues stimulate DNA binding of the p50 subunit of NF-κB. APE1 acts synergistically with low-molecular-weight reducing agents on TFs. Finally, APE1 stimulates DNA binding of the redox-insensitive p50-C62S mutant protein. Electron microscopy imaging of APE1 complexes with DNA revealed preferential polymerization of APE1 on the gapped and intrinsically curved DNA duplexes. Molecular modeling offers a structural explanation how full-length APE1 can oligomerize on DNA. In conclusion, we propose that DNA-directed APE1 oligomerization can be regarded as a substitute for diffusion of APE1 along the DNA contour to probe for anisotropic flexibility. APE1 oligomers exacerbate pre-existing distortions in DNA and enable both NIR activity and DNA binding by TFs regardless of their oxidation state.; La respiration aérobie génère des espèces réactives de l'oxygène (ERO), qui peuvent endommager les acides nucléiques, les protéines et les lipides. Un certain nombre de facteurs de transcription (TF) contiennent des résidus de cystéine sensibles à l'oxydoréduction sur leurs sites de liaison à l'ADN, c'est pourquoi l'oxydation des thiols induite par les ROS inhibe fortement leur reconnaissance des séquences d'ADN apparentées. L'endonucléase 1 apurinique /apyrimidinique (AP) humaine majeure (APE1/APEX1/HAP-1), également appelée facteur redox 1 (Ref-1), stimule les activités de liaison à l'ADN des TF oxydés tels que AP-1 et NF-κB. De plus, l'APE1 participe aux voies de réparation par excision des bases (BER) et de réparation par incision des nucléotides (NIR) pour éliminer les dommages oxydatifs des bases de l'ADN. À l'heure actuelle, le mécanisme moléculaire qui sous-tend la fonction de stimulation des TF/redox de l'APE1 et son rôle biologique restent contestés. Ici, nous apportons la preuve qu'au lieu d'une réduction directe de la cystéine dans les TF par l'APE1, les activités NIR et de stimulation des TF catalysées par l'APE1 peuvent être basées sur une liaison coopérative transitoire de l'APE1 à l'ADN et l'induction de changements conformationnels dans l'hélice. La structure du duplex de l'ADN influence fortement les activités de stimulation de la RNI et de la TF. Les endonucléases AP des plantes homologues dépourvues de résidus de cystéine conservés stimulent la liaison de l'ADN de la sous-unité p50 de NF-κB. L'APE1 agit en synergie avec des agents réducteurs de faible poids moléculaire sur les TF. Enfin, APE1 stimule la liaison de l'ADN de la protéine mutante p50-C62S insensible à l'oxydoréduction. L'imagerie au microscope électronique des complexes APE1 avec l'ADN a révélé une polymérisation préférentielle de l'APE1 sur les duplex d'ADN à espacement et à courbure intrinsèque. La modélisation moléculaire offre une explication structurelle de la façon dont l'APE1 peut s'oligomériser sur l'ADN. En conclusion, nous proposons que l'oligomérisation de l'APE1 dirigée par l'ADN peut être considérée comme un substitut à la diffusion de l'APE1 le long du contour de l'ADN pour sonder la flexibilité anisotrope. Les oligomères de l'APE1 exacerbent les distorsions préexistantes de l'ADN et permettent à la fois une activité NIR et une liaison de l'ADN par les TF, quel que soit leur état d'oxydation. Traduit avec www.DeepL.com/Translator (version gratuite)

DNA replication starts with the opening of DNA at sites called DNA replication origins. From the single sequence-specific DNA replication origin of the small Escherichia coli genome, up to thousands of origins that are necessary to replicate the large human genome, strict sequence specificity has been lost. Nevertheless, genome-wide analyses performed in the recent years, using different mapping methods, demonstrated that there are precise locations along the metazoan genome from which replication initiates. These sites contain relaxed sequence consensus and epigenetic features. There is flexibility in the choice of origins to be used during a given cell cycle, probably imposed by evolution and developmental constraints. Here, we will briefly describe their main features.

Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation., Origins of replications are associated with potential G quadruplexes forming structures (G4s). Here the authors reveal the functional role of G4 elements in DNA replication initiation.

Among other targets, the protein lysine methyltransferase PR‐Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4‐20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila , partially impairs S‐phase progression and protects from DNA re‐replication induced by stabilization of PR‐Set7. Using Epstein–Barr virus‐derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4‐20h is not sufficient to define an efficient origin per se , but rather serves as an enhancer for MCM2‐7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4‐20h‐mediated H4K20 tri‐methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1‐associated origins, which ensure proper replication timing of late‐replicating heterochromatin domains. Altogether, these results reveal Suv4‐20h‐mediated H4K20 tri‐methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes.

Spontaneous hydrolytic deamination of cytosine to uracil (U) in DNA is a constant source of genome instability in cells. This mutagenic process is greatly enhanced at high temperatures and in single-stranded DNA. If not repaired, these uracil residues give rise to C → T transitions, which are the most common spontaneous mutations occurring in living organisms and are frequently found in human tumors. In the majority of species, uracil residues are removed from DNA by specific uracil-DNA glycosylases in the base excision repair pathway. Alternatively, in certain archaeal organisms, uracil residues are eliminated by apurinic/apyrimidinic (AP) endonucleases in the nucleotide incision repair pathway. Here, we characterized the substrate specificity of the major human AP endonuclease 1, APE1, toward U in duplex DNA. APE1 cleaves oligonucleotide duplexes containing a single U⋅G base pair; this activity depends strongly on the sequence context and the base opposite to U. The apparent kinetic parameters of the reactions show that APE1 has high affinity for DNA containing U but cleaves the DNA duplex at an extremely low rate. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of a U⋅G duplex generates the expected DNA fragments containing a 5'-terminal deoxyuridine monophosphate. The fact that U in duplex DNA is recognized and cleaved by APE1 in vitro suggests that this property of the exonuclease III family of AP endonucleases is remarkably conserved from Archaea to humans. We propose that nucleotide incision repair may act as a backup pathway to base excision repair to remove uracils arising from cytosine deamination.